Compositions having fermentation analogs of virginiamycin M1 as active ingredient

ABSTRACT

Virginiamycin M 1  having the Formula: ##STR1## and virginiamycin M 1  analogs having the Formulae I-IV: ##STR2## Virginiamycin M 1  and the analogs I-IV are antagonists of cholecystokinin (CCK) and gastrin. Cholecystokinin antagonists are useful as analgesics and in the treatment and prevention of disorders of the gastrointestinal, central nervous and appetite regulatory systems in animals, especially humans. Gastrin antagonists are useful in blocking the receptors for gastrin in humans and may function as agents for the treatment of ulcers, tumors or other gastrointestinal disorders. The compounds of Formulae I-IV are also antibiotics and are useful as antimicrobial agents in animals including man and are useful as food additives to promote feed utilization in animals. Virginiamycin M 1  and the analogs of Formula I, III and IV are produced by the controlled aerobic fermentation of a strain of Streptomyces olivaceus, ATCC No. 53527, while the analog of Formula II is produced by chemical synthesis.

This is a division of application Ser. No. 934,231 filed Mar. 25, 1988,now U.S. Pat. No. 4,762,923.

OBJECTS OF THE INVENTION

It is accordingly, an object of the present invention to providevirginiamycin M₁ and analogs which are CCK and gastrin antagonists.Another object is to provide novel analogs of virginiamycin M₁ withantibiotic activity. A further object is to provide a fermentationprocess for the preparation of virginiamycin M₁ and the analogs ofFormulae I, III and IV. Another object is to provide a chemical processfor the synthesis of compounds with the Formulae I, II and III. Stillanother object is to provide pharmaceutical compositions comprisingvirginiamycin M₁ and such analogs. Another object is to provide methodsof treatment comprising administering virginiamycin M₁ and such analogsand compositions when an antibiotic, analgesic, antiulcer, antitumor ora therapeutic agent for gastrointestinal, central nervous and appetiteregulatory system disorders is indicated. These and other objects of thepresent invention will be apparent from the following description.

SUMMARY OF THE INVENTION

Virginiamycin M₁ having the Formula: ##STR3## and virginiamycin M₁analogs having the Formulae I-IV: ##STR4##

Virginiamycin M₁ and the analogs I-IV are antagonists of cholecystokinin(CCK) and gastrin. Cholecystokinin antagonists are useful as analgesicsand in the treatment and prevention of disorders of thegastrointestinal, central nervous and appetite regulatory systems inanimals, especially humans. Gastrin antagonists are useful in blockingthe receptors for gastrin in humans and may function as agents for thetreatment of ulcers, tumors or other gastrointestinal disorders. Thecompounds of Formulae I-IV are also antibiotics and are useful asantimicrobial agents in animals including man and are useful as foodadditives to promote feed utilization in animals.

BACKGROUND OF THE INVENTION

Cholecystokinin (CCK) and gastrin (G) are regulatory peptides which arefound in gastrointestinal tissue and in the central nervous system,Mutt, Gastrointestinal Hormones, Glass, Ed. Raven Press, N.Y., p. 169(1980). The CCK peptides reportedly co-exist with dopamine in certainmid-brain neurons and thus may play a role in the functioning ofdopaminergic systems in the brain, as well as serving asneurotransmitters, Prange et al., Ann. Repts. Med. Chem. 17: 31 (1982).Gastrointestinal CCK and gastrin may act on parietal and chief cells ofthe fundic glands of the mammalian gastric mucosa to stimulate acid andpepsinogen secretion, Chew and Hersey, Am. J. Physiol. 242: G504 (1982).Cholecystokinins are also believed to be physiological satiety hormonesand, thus, may play a role in appetite regulation, Smith, Eating and ItsDisorders, Stunkard and Steller, Eds., Raven Press, N.Y., p 67 (1984).Additional effects of CCKs include the stimulation of colonic motility,gall bladder concentration, pancreatic enzyme secretion and theinhibition of gastric emptying.

Intestinal CCK exists in 39- and 33-amino acid forms with the C-terminal33-amino acid residues being identical. Biological activity isrestricted to the C-terminal heptapeptide of the native peptide and aC-terminal octapeptide has the same efficacy as CCK-33 but isapproximately ten-times more potent, Jensen et al., Proc. Natl. Acad.Sci. USA 77: 2079 (1980).

Gastrin also occurs naturally in several different forms; 34-aminoacids, 17-amino acids and 13-amino acids with the tyrosine being eithersulfated or unsulfated. The 17- and 13-amino acid forms can be viewed asC-terminal fragments of the 34-amino acid form. The different formsexhibit varying potency for stimulating gastric acid secretion.Gastrin-17 is 5 times more potent than Gastrin-34 and 2.5 times morepotent than Gastrin-13, Walsh and Grossman, New Engl. J. Med. 292:1324,1377 (1975). Gastrin and CCK share a common C-terminal pentapeptideamide sequence, Gly--Trp--Met--Asp--Phe--NH₂.

Gastrin and CCK antagonists are useful for treating diseases mediated bygastrin and CCK. CCK antagonists are successful in preventing ortreating CCK-related disorders of the gastrointestinal, central nervousand appetite regulatory systems of animals, especially of humans.Antagonists of CCK are also useful in potentiating and prolongingopiate-mediated analgesia and thus have utility in the treatment ofpain, Faris et al., Science 226: 1215 (1984). Gastrin antagonists areuseful in the treatment and prevention of gastrin-related disorders ofthe gastrointestinal system in humans and animals, such as ulcers,Zollinger-Ellison syndrome, antral G cell hyperplasia and otherconditions, in which reduced gastrin activity is of therapeutic value.CCK and gastrin also have trophic effects on certain tumors, Ohyama,Hokkaido J. Med. Sci., 60, 206 (1985), and antagonists of CCK andgastrin are useful in treating these tumors.

Antibiotics of the Virginiamycin family have been used as food additivesto improve the growth of poultry, swine and cattle. Although growthpromotion by antibiotics is not thoroughly understood, there is littledoubt that the effect is due in part to an inhibition of the intestinalflora, Coccito, Micro. Rev. 43: 145 (1979). Virginiamycin M₁ and relatedantibiotics are generally specific for gram-positive bacteria andprevent cell multiplication. The virginiamycin antibiotics are mosteffective when in their complex form, that is when they are composed ofboth M and S components, Coccito, Micro. Rev. 43: 145 (1979). The wideuse of these antibiotics as growth promoters relates their low toxicity,lack of accumulation in animal tissues, no production of resistantmutants and a rapid degradation in animal feces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a H¹ -nuclear magnetic resonance (NMR) spectrum of thecompound of Formula I.

FIG. 2 is a H¹ -nuclear magnetic resonance (NMR) spectrum of thecompound of Formula II.

FIG. 3 is a H¹ -nuclear magnetic resonance (NMR) spectrum of thecompound of Formula III.

FIG. 4 is a H¹ -nuclear magnetic resonance (NMR) spectrum of thecompound of Formula IV.

DETAILED DESCRIPTION OF THE INVENTION

Virginiamycin M₁ and the compounds of Formulae I, III and IV areproduced by controlled aerobic fermentation of a strain of theStreptomyces olivaceus. It is to be understood that for the fermentationproduction of virginiamycin M₁ and the compounds of Formulae I, III andIV the present invention is not limited to the use of this specificspecies or strain of Streptomyces. It is especially desired and intendedthat there be included within the scope of this invention, the use ofother natural or artificial mutants produced or derived from thedescribed cultures, or other variants or species of the genusStreptomyces in so far as they can produce virginiamycin M₁ and thecompounds of Formulae I, III and IV or any related analogs. Theartificial production of mutant species or strains of this strain ofStrentomyces may be achieved by conventional physical or chemicalmutagens, for example, ultraviolet irradiation of the describedcultures, or nitrosoguanidine treatment and the like. Recent recombinantDNA techniques such as plasmid incorporation in bacteria and fungi andthe like may also prove useful.

Virginiamycin M₁ and the compounds of Formulae I, III and IV areproduced by the controlled aerobic fermentation of a species ofStreptomyces which has been deposited in the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., on July29, 1986, under the "Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure" andhas been assigned ATCC number 53527.

The Streptomyces species was isolated from soil associated with treeroots in Pimpri, India and expresses morphological and culturalcharacteristics as shown in the following table:

TABLE I

Morophology: Sporophores form very loose coils, chains are more than 15spores in length, spores are spherical to oval, and are about 0.9μ to0.9×1.2μin size

Cultural Charastics: Yeast extract-malt extract agar (ISP Medium 2)

V: Reverse - grayish brown edged with gray

A. Light gray mixed with white and edged with dark gray

SP: None

Oatmeal agar (ISP Medium 3)

V: Reverse - light grayish tan edged with gray

A: Light gray mixed with white, edged with dark gray

SP: None

Inorganic salts-starch agar (ISP Medium 4)

V: Reverse - grayish tan edged with gray

A: Light gray mixed with white, edged with dark gray

SP: None

Glycerol asparagine agar (ISP Medium 5)

V Reverse - tan edged with gray

A: Light gray and white edged with light gray

SP: None

Peptone-iron-yeast extract agar (ISP Medium 6)

V: Reverse - tan

A: Pale gray mixed with white and edged with medium gray

SP: None

Melanin: None

Tyrosine Agar (ISP Medium 7)

V: Reverse - tan edged with brown

A: Mixed grayish - white and gray edged with dark gray

SP: Slight browning of medium

Czapek-Dox Agar

V: Reverse - grayish tan

A: Light gray mixed with white

SP: Slight browning of medium

V=vegetative growth; A=aerial mycelium; SP=soluble pigment

Color number designations taken from Color Harmony Manual, 1958, 4thEdition, Container Corporation of America, Chicago, Ill.

The carbon utilization of ATCC 53527 was determined usingPridham-Gottlieb basal medium (ISP Medium 9) supplemental with 1% carbonsources as seen in the following table:

                  TABLE II                                                        ______________________________________                                        Glucose      +         Mannitol     +                                         Arabinose    +         Mannose      +                                         Cellulose    -         Raffinose    ±                                      Fructose     +         Rhamnose     +                                         Inositol     +         Sucrose      +                                         Lactose      +         Xylose       +                                         Maltose      +                                                                ______________________________________                                         [+ = growth; ± = growth poor or questionable; - = no growth as compare     to negative control (no carbon source)                                   

The temperature growth range and oxygen requirements were determinedusing yeast extract-dextrose+salts agar. The results are shown in thefollowing table:

                  TABLE III                                                       ______________________________________                                        Temperature range (Yeast extract-dextrose + salts agar)                       ______________________________________                                        28° C.- Good vegetative growth and                                                    sporulation                                                    37° C.- Good vegetative growth and                                                    sporulation                                                    42° C.- No growth                                                      50° C.- No growth                                                      ______________________________________                                         Oxygen requirements (Stab culture in yeast extractdextrose + salts agar) 

Aerobic

All readings taken after three weeks at 28° C. unless noted otherwise.pH of all media approximately neutral (6.8-7.2).

The cultural characteristics of this organism are compared with culturedescriptions of Streptomyces species described in Berger's Manual ofDeterminative Bacteriology, Eighth Edition, 1974, Williams & Wilkens,Baltimore, Md., and the Streptomeyces species is presumptivelyidentified as a strain of Streptomyces olivaceus.

The fermentation is carried out at a temperature range of from about 20°C. to about 37° C., with 28° C. being preferred. Generally, thecomposition of the assimilable nutrient medium may be varied over a widerange. The essential nutrient ingredients are a carbon source and anitrogen source. Other essential nutrients are provided via mineralsalts such as chlorides, nitrates, sulfates, carbonates and phosphatesof sodium, potassium, ammonium and calcium. The nutrient medium may alsocontain sources of inorganic trace elements such as magnesium, iron,copper, manganese, zinc, cobalt and the like.

Typical sources of carbon include; sugars, oils, organic acids, dextrin,starches, glycerol and the like. Typical nitrogen sources include; aminoacids, vegetable meals, and extracts (e.g., malts, soy, cotton seed,figs, tomato, corn, etc.), animal viscera, various hydrolysates (e.g.,casein, yeast, etc.) and industrial by-products such as lard water anddistillers solubles.

The maximum yield of virginiamycin M₁ and the compounds of Formulae I,III and IV is achieved within about 24 to about 96 hours, usually inabout 48 to about 72 hours, of fermentation under optimum conditions.The inoculum for the fermentation is provided from vegetative growth ina medium which supports rapid growth of the microorganism or by directinoculation of frozen biomass.

The terms "seed" and "production" media are employed as terms of art.Generally, a seed medium supports rapid growth of the microorganism andan aliquot (seed) of this medium is used to inoculate a productionmedium for a large scale fermentation.

Following fermentation, the accumulated virginiamycin M₁ and thecompounds of Formulae I, III and IV are recovered from the culture brothby conventional chromatographic means. The individual compounds areseparated by reverse phase high performance chromatography.

Virginiamycin M₁ and the compounds of Formulae I, III and IV areisolated from whole fermentation broth by the addition of a nearly equalvolume of a moderately polar solvent immiscible in water. Such solventsinclude chloroform, ethyl acetate, methyl ethyl ketone and the like,with methyl ethyl ketone being preferred. The layers are allowed tosettle and the organic layer collected. The organic layer containsvirginiamycin M₁ and the compounds of Formulae I, III and IV.

Isolation of the various compounds from the fermentation broth is basedon specific biological activity. The products resulting from thechemical conversion of virginiamycin M₁ to compound III and subsequentlyto compounds I and II are also assayed for biological activity. Thebiological assays include gastrin binding to mammalian gastric glands,preferably guinea pig and CCK binding to mammalian brain, preferablyguinea pig or mammalian pancreas, preferably rat.

The active organic layer isolated from the culture broth was flashevaporated under reduced pressure at a temperature of about 30° C. toabout 50° C., with 40° C. being preferred, and partitioned between asaturated hydrocarbon such as hexane and an alcohol such as methanol.The methanol layer is flash evaporated at about 30° C. to about 50° C.,with 40° C. being preferred, under pressure and chromatographed onsilica gel using a mobile phase of acetone about 50% to about 75% inhexane. The active fraction was purified by reverse phase highperformance liquid chromatography (HPLC).

Virginiamycin M₁ is converted to the compound of Formula III by theaddition of about 1 mole equivalent of methanesulfonyl chloride to ananhydrous pyridine solution of virginiamycin M₁. The mesylation reactionis allowed to proceed at about 25° C. for about 20 minutes and thepyridine is removed by flash evaporation. The residue is partitioned inabout 1% aqueous NaCl and dichloromethane. The organic layer is thendried and chromatographed by reverse phase HPLC. The compound of FormulaIII is reduced to the compounds of Formulae II and I by sodiumborohydride reduction. To a methanolic solution of the compound ofFormula III is added a slight excess of 1 mole equivalent of a NaBH₄/methanol solution at about 10 mg/ml. Purification is similar to thatfor the fermentation products.

Biological activity of virginiamycin M₁ and the compounds of Formulae I,II, III and IV is determined by gastrin receptor binding, pancreas CCKreceptor binding, brain CCK binding and microbial growth inhibitionassays.

Virginiamycin M₁ and the compounds of Formulae I, II, III and IV can beadministered to mammals, including humans, either alone, or preferably,in combination with pharmaceutically acceptable carriers or diluents, ina pharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally.Parenteral administration includes intravenous, intramuscular,intraperitoneal, subcutaneous, and topical administration.

For oral use as CCK or gastrin antagonists or as antibiotics, theselected compound can be administered, for example, in the form oftablets or capsules, or as an aqueous solution or suspension. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch, and lubricating agents, such as magnesiumstearate, are commonly added. For oral administration in capsule form,useful diluents are lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. For oral use as antibiotics indomestic animals, the compounds of Formulae I, II, III, and IV are addedto the animal feed. If desired, certain sweetening and/or flavoringagents can be added. For intramuscular, intraperitoneal, subcutaneous,and intravenous use, sterile solutions of the active ingredient areusually prepared, and the pH of the solutions should be suitablyadjusted and buffered. For intravenous use, the total concentration ofsolutes should be controlled to render the preparation isotonic.

When virginiamycin M₁ and the compounds of Formulae I, II, III, and IVare used as a CCK or gastrin antagonist or an antibiotic in a humansubject, the daily dosage will normally be determined by the prescribingphysician. Moreover, the dosage will vary according to the age, weight,and response of the individual patient, as well as the severity of thepatient's symptoms. However, in most instances, an effective dailydosage will be in the range from about 1 mg to about 1500 mg andpreferably 10 mg to 500 mg in a single or divided doses. On the otherhand, it may be necessary to use dosages outside these limits in somecases.

When the compounds of Formulae I, II, III, and IV are used as an oralantibiotics for domestic animals, the daily dosage will be determinedaccording to the age, weight, and species of animal being treated. Theeffective dosage will be in the range from about 5 to about 200 partsper million per volume of food.

Virginiamycin M₁ and the compounds of Formulae I, II, III and IV bind toboth CCK and gastrin receptors. Thus, virginiamycin M₁ and the fouranalogues may have a unique function in appetite regulation, thetreatment of CCK-related disorders of the gastrointestinal and centralnervous system. The unique action of virginiamycin M₁ and the fouranalogues as gastrin antagonists will allow their use in the treatmentand prevention of gastrin-related disorders such as ulcers,Zollinger-Ellison syndrome and antral G cell hyperplasia. The fouranalogs also function as antibiotic agents and can be used to treatmicrobial infections.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

Fermentation

A lyophilized tube of the Streptomyces ATCC No. 53527 was added underaseptic conditions to 54 ml of a seed medium in a 250 ml baffledErlenmeyer flask. The seed media contained the following: dextrose, 1gm/L; soluble starch, 10 g/L; beef extract, 3 g/L; ardamine pH, 5 g/L;NZ amine, 5 g/L; MgSO₄ ·7H₂ O, 0.08 g/L; phosphate buffer, 2 ml/L andCaCO₃, 0.5 g/L. The pH of the medium was between 7.0 and 7.2. The flaskwas maintained at 28° C. on a 220 rpm rotary shaker for 48 hours. Ten mlof the 48 hour culture was aseptically transferred to 500 ml in a 2 Lbaffled flask and incubated at 28° C. on a 220 rpm rotary shaker for 24hours. A 500 ml inoculum from the second seed culture was added to 10 Lof production medium in a 14 L fermenter. The production media containedcorn gluten meal, 5.0 g/L; primatone HS, 2.5 g/L; yeast extract (Fidco),1.0 g/L; malt extract, 10.0 g/L; sucrose, 5.0 g/L; CaCO₃, 5.0 g/L andP-2000, 2.0 ml. The pH of the production medium was between 7.2 and 7.4.The inoculated production media was incubated for 64 hours at 28° C.,aerated at a flow of 3 L per minute and agitated at 400 rpm.

EXAMPLE 2

Isolation and Characterization of Virginiamycin M₁ and Compounds of theFormulae I, III and IV

Forty L of whole culture broth from Example 1 was extracted with 48 L ofmethylethyl ketone. The organic layer contained the biological activityand was flash evaporated under reduced pressure at 40° C. to give aresidue. The residue was partitioned between hexane, 1 L, and methanol,1 L, with the hexane layer being discarded. The active methanol layerwas flash evaporated under reduced pressure at 40° C. to yield a 50 gdry weight sample. Flash chromatography of this material in 5 runs on500 g silica gel 60 (4 μm) using 50% acetone/hexane and then 75%acetone/hexane as the mobile phases yielded active fractions with atotal dry weight of 3 grams. Subsequent preparative reverse phase highperformance liquid chromatography of this material in 3 runs on a ZorbaxC-8, 2.12×25 cm, column using 40% acetonitrile/water as the isocraticmobile phase at room temperature and 15 ml/min yielded 92 mg ofvirginiamycin M₁ ; 5.2 mg of the compound of Formula I; 16.4 mg of thecompound of Formula III and 25.9 mg of the compound of Formula IV.

Physiochemical characterization of the virginiamycin M₁ isolated fromthe present fermentation was identified by comparison with EI-MS and ¹H--NMR data (M+=525) from the literature, Kingston et al., J. Chem. Soc.19: 1669-1676 (1966), and an authentic sample obtained by purifying acommercially available virginiamycin. Subsequent biological/biochemicaltesting on virginiamycin M₁ was performed on this authentic sample. Bothsamples showed identical HPLC retention times and UV spectra (λ_(max)=210.5 nm; E %=535). A 300 MHz H¹ -H¹ homonuclear correlation NMRspectrum was recorded for the first time for characterization.

High resolution mass spectral data of the compound of Formula I revealeda molecular formula of C₂₈ H₃₅ N₃ O₆ (calculated: 509.2526; found:509.2528). Its 300 MHz H¹ --NMR spectrum, see FIG. 1, in CD₂ Cl₂ furthercorroborated the purity and identity of this compound. Broad banddecoupled C¹³ --NMR spectrum in CD₂ Cl₂ revealed resonances at 12.11;18.82; 19.77; 20.34; 29.91; 30.13; 36.77 38.87; 42.26; 51.08; 70.93;82.46; 123.77; 123.85; 125.78; 126.46; 127.11; 127.72; 129.20; 132.85;132.93; 135.20; 137.13; 143.29; 143.58; 161.37; 161.86; 167.13 ppm. Itsultraviolet spectrum in methanol displayed absorption maxima at 212 nm(E %=557) and 274.5 nm (E %=764). Its infrared spectrum in CH₂ Cl₂solution showed absorption bands at 3595; 3375; 1730; 1670 and 1620cm⁻¹. The natural and semisynthetic compound, Example 4, showedidentical mass, H¹ --NMR, and UV spectra, and HPLC retention time andallowed the assignment of the following structure: ##STR5##

High resolution mass spectral data for the compound of Formula IIIrevealed a molecular formula of C₂₈ H₃₃ N₃ O₆ (calculated: 507.2369;found: 507.2369). Its 300 MHz H¹ --NMR spectrum, see FIG. 3, in CD₂ Cl₂further corroborated the purity and identity of this product. Its broadband decoupled C¹³ --NMR spectrum in CD₂ Cl₂ revealed resonances at12.19; 18.78; 1960; 21.37; 30.10; 30.22; 37.60; 40.65; 42.04; 52.33;81.18; 125.38; 126.56; 126.79; 127.25; 129.46; 131.85; 136.98; 142.91;143.20; 143.32; 146.19; 160.28; 160.54; 167.12; and 192.78 ppm. Itsultraviolet spectrum in methanol displayed absorption maxima at 212 nm(E %=545.5) and 325 nm (E %=417). Its infrared spectrum in CH₂ Cl₂solution showed absorption bands at 3380; 1730; 1662 and 1625 cm⁻¹. Thenatural and semisynthetic compound, Example 3, showed identical mass, H¹--NMR, and UV spectra, and HPLC retention time and allowed theassignment of the following structure: ##STR6##

High resolution mass spectral data for the compound of Formula IV on thebis-trimethylsilyl derivative revealed a molecular formula of C₂₈ H₃₅ N₃O₆ +T₂ (calculated 653.3316; found 653.3452). Its 300 MHz H'--NMRspectrum, see FIG. 4, in CD₂ Cl₂ further corroborated the purity andidentity of this product. Its ultraviolet spectrum in methanol is almostidentical to that of Virginiamycin M₁ and displayed an absorptionmaximum at 213 nm (E %=542) and allowed the assignment of the followingstructure: ##STR7##

EXAMPLE 3

Synthesis of the Compound of Formula III

Virginiamycin M₁, from Example 2 or obtained commercially, was convertedto the compound of Formula III by adding methanesulfonyl chloride, 0.2ml, to 199.36 mg of virginiamycin M₁ in anhydrous pyridine solution, 5ml. The mesylation reaction was allowed to proceed at room temperaturefor 20 minutes. Flash evaporation of the solvent, pyridine, underreduced pressure and at 40° C. gave a reidue. Partition of this residuein 1% aqueous NaCl, 20 ml, and dichloromethane (2×25 ml) followed bydrying the organic layers in anhydrous Na₂ SO₄ and flash removal ofsolvent under reduced pressure and at 40° C. yielded 245 mg of a crudeproduct. Analytical high performance liquid chromatography of thisproduct revealed the presence of compound III as the predominant productand the absence of virginiamycin M₁. Preparative reverse phasechromatography of the crude product on a Zorbax ODS column with 40%aqueous MeCN as the mobile phase at 15 ml/minutes provided 88.15 mg ofpure compound III.

EXAMPLE 4

Synthesis of Compounds of the Formulae I and II

Compound III was converted to compounds of the Formulae I and II. To amethanolic solution, 10 ml of compound III, from Example 1 or Example 3,was added 0.5 ml of a 10 mg/ml NaBH₄ /MeOH solution. High pressureliquid chromatographic analysis of the reaction mixture revealed thatthe reaction was immediate and the products were compound I and itsepimer, compound II. The reaction was then worked up. The addition ofacetone, 0.1 ml, to the reaction mixture destroyed any excess reagent.Partition of the mixture between 1% aqueous NaCl, 50 ml, and CH₂ Cl₂,2×50 ml, followed by drying the organic layers in anhydrous Na₂ SO₄ andflash evaporation under reduced pressure at 30° C. afforded 54.8 mgmaterial. Purification of this material on a preparative Zorbax ODScolumn, 2.12×25 cm, with 35% aqueous MeCN as the isocratic mobile phaseat 15 ml/min at room temperature yielded 27.69 mg of Compound I and 15.7mg of its epimer, compound of the Formula II.

The physiochemical characterization of the compound of Formula I ispresented in Example 2.

High resolution mass spectral data for the compound of Formula IIrevealed a molecular formula of C₂₈ H₃₅ N₃ O₆ (calculated: 509.2526;found: 509.2528). Its 300 MHz H¹ --NMR spectrum, see FIG. 2, in CD₂ Cl₂further corroborated the purity and identity of this compound. Its broadband decoupled C¹³ --NMR spectrum in CD₂ Cl₂ revealed resonances at12.03; 18.83; 19.67; 20.47; 30.07; 35.95; 39.10; 42.06; 51.04; 69.88;81.75; 124.39; 124.47; 125.52; 126.98; 127.51; 127.57; 129.60; 129.62;132.61; 135.19; 137.19; 143.22; 143.50; 161.23; 161.90 and 167.04 ppm.Its ultraviolet spectrum in methanol displayed absorption maxima at 212nm (E %=522) and 274.5 nm (E %=701). Its infrared spectrum in CH₂ CL₂solution showed absorption bands at 3600, 3375; 1730; 1670 and 1625cm⁻¹. These characteristics allowed the assignment of the followingstructure: ##STR8##

EXAMPLE 5

Gastrin Receptor Binding in Guinea Pig Gastric Glands

Guinea pig gastric mucosal glands were prepared by the procedure ofPraissman et al., J. Recentor Res. 3:647 (1983). Stomachs from maleHartley guinea pigs (250-400 g body weight) were washed thoroughly andminced with fine scissors in HEPES buffer consisting of the following:130 mM NaCl, 12 mM NaHCO₃, 3 mM NaH₂ PO₄, 3 mM Na₂ HPO₄, 3 mM K₂ HPO₄, 2mM MgSO₄, 1 mM CaCl₂, 5 mM glucose and 4 mM L-glutamine, 25 mM HEPES atpH 7.4. The minced tissues were washed and incubated in a 37° C. shakerbath for 40 minutes, with HEPES buffer containing 0.1% collagenase and0.1% bovine serum albumin (BSA), and bubbled with 95% O₂ and 5% CO₂. Thetissues were passed twice through a 5 ml glass syringe to liberate thegastric glands, and then filtered through 200 mesh nylon. The filteredglands were centrifuged at 270×g for 5 minutes and washed twice byresuspension and centrifugation.

The washed guinea pig gastric glands were resuspended in 25 ml of HEPESbuffer containing 0.25 mg/ml of bacitracin. For binding studies, 14.2 μlof 50% MeOH/H₂ O (for total binding) or gastrin (1 μM finalconcentration, for nonspecific binding) or broth or test compound fromExamples 2, 3 or 4 and 20 μl of ¹²⁵ I-gastrin (New England Nuclear[NEN], 20,000 cpm 20 μl ) were added to 220 μl of gastric glands intriplicate tubes which were aerated with 95% O₂ and 5% CO₂ and caped.The reaction mixtures, after incubation at 25° C. for 30 minutes in ashaking water bath, were filtered under reduced pressure on glass GF/Bfilters (Whatman) and immediately washed with 3×4 ml of HEPES buffercontaining 0.1% BSA fraction V. The radioactivity on the filters wasmeasured using a Beckman 5500 gamma counter for ¹²⁵ I-gastrin.

The results of the gastrin binding assay are shown in the followingtable:

                  TABLE IV                                                        ______________________________________                                        Gastrin Receptor Binding                                                      Compound        IC.sub.50 (nM)                                                ______________________________________                                        Virginiamycin M.sub.1                                                                         710                                                           I               12                                                            II              13                                                            III             90                                                            IV              360                                                           ______________________________________                                    

EXAMPLE 6

Pancreatic Cholecystokinin Receptor Binding Assay

Radiolabeled CCK-8 was purchased from New England Nullear as ¹²⁵I-CCK-8. Receptor binding was performed according to Innis and SnyderProc. Natl. Acad. Sci., 77, 6917 (1980), with the minor modification ofadding the additional protease inhibitors, phenylmethane sulfonylfluoride (PMSF) and o-phenanthroline, which have no effect on the ¹²⁵I-CCK receptor binding assay. All assays were performed in triplicate.Pancreatic receptor membranes were prepared by homogenzing a gram offresh rat pancreas tissue in 30 ml TRIS-HCl (50 mM), pH 7.7 using aBrinkman polytron PT10 (Brinkman). The homogenate was washed twice andcollected by centrifugation at 48,000×g at 4° C. for 10 minutes. Themembranes were resuspended in assay buffer consisting of 5 mMdithiothieitol, 0.1 mM bacitiacin, 5 mM MgCl₂ ·H₂ O, 0.2% heat denaturedBSA, 1.2 mM PMSF and 0.5 mM O-phenanthroline at a concentration of 100ml per gram of pancreas. To each incubation tube, in an ice-bath,containing 0.45 ml of membrane preparation, 25 μl of assay buffer (fortotal binding) or unlabeled CCK 26-33 (for non-specific binding) orbroth or test compound from Examples 2, 3 or 4 for determining theinhibition of specific CCK binding by broths, and 25 μl of ¹²⁵ I-CCKwere added. The reaction mixtures were mixed briefly and placed in a 37°C. water bath with gentle shaking for 30 minutes. The reaction mixtureswere then diluted with 4 ml of ice-cold TRIS buffer, pH 7.7, containing1 mg/ml bovine serum albumin and immediately filtered in Watman G F/Bfilters. The filters were washed with 3×4 ml of the same buffer and theradioactivity associated with the filters was counted in a Beckman Gamma5500 counter. Broths were prepared by adding two volumes of acetone tooriginal broth; supernatants were freeze dried after centrifugation at1500 g for 10 minutes. The dried samples were then brought up inmethanol/H₂ O (1:1) and ready for assay.

The results of the pancreatic CCK binding assay are shown in thefollowing table:

                  TABLE V                                                         ______________________________________                                        Pancreatic CCK Receptor Binding                                               Compound              IC.sub.50 (nM)                                          ______________________________________                                        Virginiamycin M.sub.1 >100,000                                                I                     >10,000                                                 II                    >10,000                                                 III                   >10,000                                                 ______________________________________                                    

EXAMPLE 7

Brain Cholecystokinin Receptor Bindinc Assay

CCK-8 binding was performed according to the description for thepancreatic method, with modifications by Saito et al., J. Neurochem.,37, 483 (1981).

The brains from male Hartley guinea pigs (300-500 g) were removed andplaced in ice-cold 50 mM Tris HCl plus 7.58 g/L Trizma-7.4 [pH 7.4 at25° C.]. The cerebral cortex was dissected and used as a receptor sourceand each gram of fresh guinea pig brain tissue was homogenized in 10 mlof Tris/Trizma buffer, pH 7.4, with a Brinkmann polytron PT-10. Thehomogenates were centrifuged at 42,000×g for 15 minutes, then theresulting pellets were resuspended in 200 volumes of binding assaybuffer (10 mM N-2-hydroxy-ethyl-piperazine-N'-2-ethane-sulfonic acid(HEPES), pH 7.7 at 25° C., mM MgCl₂, 1 mM ethyleneglycol-bis-(β-amino-ethyl-ether-N,N'-tetraacetic acid (EGTA), 0.4% BSAand 0.25 mg/ml bacitracin, pH 6.5).

The remainder of the binding assay method was as described for thepancreatic method, Example 6, except that the reaction mixtures wereincubated at 25° C. for 2 hours before filtration and the filters werewashed with HEPES buffer.

The results of the brain CCK binding assay are shown in the followingtable:

                  TABLE VI                                                        ______________________________________                                        Brain CCK Receptor Binding                                                    Compound        IC.sub.50 (nM)                                                ______________________________________                                        Virginiamycin M.sub.1                                                                         571                                                           I               7.8                                                           II              6.1                                                           III             13.3                                                          IV              270                                                           ______________________________________                                    

EXAMPLE 8

Antibiotic Activity

The compounds of Formulae I, II, and IV and virginiamycin M₁ fromExample 2 were assayed for antibiotic activity following a methoddescribed by Matsen and Barry, Manual of Clinical Microbiology,Lennette, Spaulding, and Truant, Eds., American Society forMicrobiology, Washington, D.C., p. 418 (1974). The minimal inhibitoryconcentrations (MIC) were determined by applying serial dilutions of thecompounds of Formulae I, III, and IV and virginiamycin M₁ to standardfilter paper discs. The concentration of the test compounds ranged from0.05 μg to 3.0 μg per disc in 20 μl of diluent. The test organism,Micrococcus luteus, was innoculated on the surface of an agar basednutrient medium in petri plates and the discs placed on the surface ofthe medium. The nutrient media consisted of nutrient broth supplementedwith 0.2% yeast extract and 1.5% agar. The innoculated plates wereincubated for 15 hours at 28° C. and the zones of inhibition determined.The MIC of each compound was determined by the method described inMatsen and Barry, supra. The results are shown in the following table:

                  TABLE IV                                                        ______________________________________                                        Minimal Inhibitory Concentration                                              Compound        MIC (μM)                                                   ______________________________________                                        Virginiamycin M.sub.1                                                                         1.5                                                           I               90.0                                                          III             250.0                                                         IV              15.0                                                          ______________________________________                                    

What is claimed is:
 1. A pharmaceutical composition useful for treatinggastrointestinal disorders, central nervous system disorders andregulating appetite in mammals comprising a pharmaceutically effectiveamount of virginiamycin M₁ and an acceptable pharmaceutical carrier. 2.A pharmaceutical composition useful for treating gastrointestinaldisorders, central nervous system disorders and regulating appetite inmammals comprising a pharmaceutically effective amount of the compoundof Formula I ##STR9## and an acceptable pharmaceutical carrier.
 3. Apharmaceutical composition useful for treating gastrointestinaldisorders, central nervous system disorders and regulating appetite inmammals comprising a pharmaceutically effective amount of the compoundof Formula III ##STR10## and an acceptable pharmaceutical carrier.
 4. Apharmaceutical composition useful for treating gastrointestinaldisorders, central nervous system disorders and regulating appetite inmammals comprising a pharmaceutically effective amount of the compoundof Formula IV ##STR11## and an acceptable pharmaceutical carrier.
 5. Apharmaceutical composition useful for treating bacterial infections inmammals comprising a pharmaceutically effective amount of the compoundof Formula I ##STR12##
 6. A pharmaceutical composition useful fortreating bacterial infections in mammals comprising a pharmaceuticallyeffective amount of the compound of Formula III ##STR13##
 7. Apharmaceutical composition useful for treating bacterial infections inmammals comprising a pharmaceutically effective amount of the compoundof Formula IV ##STR14##
 8. A pharmaceutical composition useful fortreating bacterial infections in mammals comprising a pharmaceuticallyeffective amount of the compounds of Formula I ##STR15##
 9. Apharmaceutical composition useful for treating bacterial infections inmammals comprising a pharmaceutically effective amount of virginiamycinM₁ and the compounds of Formula I ##STR16##